Distinct inter-domain interactions of dimeric versus monomeric α-catenin link cell junctions to filaments

Attachment between cells is crucial for almost all aspects of the life of cells. These inter-cell adhesions are mediated by the binding of transmembrane cadherin receptors of one cell to cadherins of a neighboring cell. Inside the cell, cadherin binds β-catenin, which interacts with α-catenin. The transitioning of cells between migration and adhesion is modulated by α-catenin, which links cell junctions and the plasma membrane to the actin cytoskeleton. At cell junctions, a single β-catenin/α-catenin heterodimer slips along filamentous actin in the direction of cytoskeletal tension which unfolds clustered heterodimers to form catch bonds with F-actin. Outside cell junctions, α-catenin dimerizes and links the plasma membrane to F-actin. Under cytoskeletal tension, α-catenin unfolds and forms an asymmetric catch bond with F-actin. To understand the mechanism of this important α-catenin function, we determined the 2.7 Å cryogenic electron microscopy (cryoEM) structures of filamentous actin alone and bound to human dimeric α-catenin. Our structures provide mechanistic insights into the role of the α-catenin interdomain interactions in directing α-catenin function and suggest a bivalent mechanism. Further, our cryoEM structure of human monomeric α-catenin provides mechanistic insights into α-catenin autoinhibition. Collectively, our structures capture the initial α-catenin interaction with F-actin before the sensing of force, which is a crucial event in cell adhesion and human disease.

. CryoEM data processing workflow for full-length human monomeric acatenin A representative image of the patch motion-corrected and patch CTF estimated micrograph is shown at the top. The scale bar on the micrograph corresponds to 50 nm. Representative 2D class averages of the particles used for structure determination are provided. Thumbnail images for various 3D volumes obtained at different stages are shown. The number of particles and resolution computed using two half-maps using a gold-standard Fourier shell correlation cut-off value of 0.143 are indicated. The 3D reconstruction of monomeric a-catenin resulted in a 6.9 Å resolution map.

Figure S2. The FABD of monomeric a-catenin engages in new interdomain interactions
a Ca trace of our cryoEM structure of monomeric a-catenin. The middle domain is colored from black to white for residues 277 to 668. The carboxy-terminal F-actin binding domain (FABD) is colored spectrally from shorter to longer wavelengths as indicated for residue 641 to 861. b Ca trace of subunit 'A' of the crystal structure of dimeric a-catenin (PDB entry 4igg) 1 , color-coded and oriented as in panel (A). The amino-terminal domain is not shown. c Ca trace of subunit 'B' of the crystal structure of dimeric a-catenin (PDB entry 4igg) 1 , color-coded and oriented as in panel (A). The amino-terminal domain is not shown. d New monomeric a-catenin interdomain interactions. The side chain representation is merely to show the characteristics of regions and their overall domain interaction and requires further confirmation through alternate approaches.

Figure S3. CryoEM data processing workflow for unbound F-actin and F-actin bound by dimeric a-catenin
Representative micrographs are shown before (top, left) and after particle picking (top, right; red traces). Representative 2D classes of filaments (unbound and decorated, respectively) for F-actin bound by dimeric a-catenin are shown with a box size of 603 Å. The final 3D reconstruction workflow was the same for both maps resulting in a 2.7 Å resolution map for both unbound F-actin and F-actin bound by dimeric a-catenin, as estimated in cryoSPARC by Fourier shell correlation (FSC) with a cutoff at 0.143. The EMDB deposition identifiers, the fitted structures, and the corresponding PDB deposition identifiers are provided. Figure S4. The 2.8 Å cryoEM structure of dimeric a-catenin bound to F-actin (Left) The refined coordinates of dimeric a-catenin bound to F-actin are overlayed onto the final 3D reconstruction (transparent grey surface). The actin subunits are colored brown while the F-actin binding domain of the dimeric a-catenin is colored red, green, or blue. (Right) The structure of a-catenin bound to F-actin looking onto the carboxy-terminal interaction of the a-catenin FABD (in green) with an adjacent a-catenin FABD (in blue) overlaid onto the cryoEM map (transparent grey surface). The actin subunits are shown semi-transparent for clarity.

Figure S5. The carboxy-terminus of the FABD of dimeric a-catenin engages in new interdomain interactions
The carboxy-terminus (red) of the FABD interacting with the second a-helix (blue) of the 5-helix FABD bundle as seen in (a) subunit 'A' and (b) subunit 'B' of our dimeric a-catenin crystal structure (PDB entry 4igg) 1 or (c) in our F-actin bound dimeric a-catenin cryoEM structure. The orientation is the same in panels (a) through (c). Note the unique interaction of N710 residing at the amino terminus of the second a-helix (blue). d Ca trace of (left) subunit 'B' of our crystal structure of dimeric a-catenin (PDB entry 4igg) 1 or of (right) our F-actin bound dimeric cryoEM a-catenin structure. The a-catenin FABD is colored spectrally as indicated. The first a-helix (right; 'H1') of the FABD in the unbound a-catenin structure is a disordered amino-terminal of residue 710 in the actinbound structure (left) whereby the 860 region (arrow) fills the space of the first a-helix. e Superposition Ca trace of (left) subunit 'A' or of (right) subunit 'B' of our crystal structure of dimeric a-catenin (PDB entry 4igg) 1 onto our F-actin bound dimeric cryoEM a-catenin structure. Note the movement (arrows) induced by the binding of F-actin for residues 796 (5.8 Å for 'A' and 8.6 Å for 'B') and 816 (4.7 Å for 'A' and 5 Å for 'B').

Table S1. Absolute molar mass determination by size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS)
Individual peak fractions corresponding to human a-catenin monomer or dimer were pooled from previous size exclusion chromatography (SEC) runs and used for SEC-MALS analyses. The absolute molar mass was analyzed using Astra 6.0. a The dimeric human a-catenin SEC pool has 13.8% of monomeric a-catenin reappearing on the SEC-MALS run. b The monomeric human a-catenin SEC pool has 7.9% of dimeric a-catenin reappearing on the SEC-MALS run. c The dimeric human a-catenin D1-21 SEC pool remains a dimer on the SEC-MALS run.